Microbial pathogens may infect their host through one of several mechanisms. These microbial pathogens may enter their host through wounds on the skin, or via interaction with mucosal surfaces. Bacterial pathogens and viral pathogens which affect the host through this mechanism first come in contact with a mucosal surface, adhere thereto, and then colonize thereon, or are taken up by specialized absorptive cells (M cells) which exist inside the epithelium over Peyer's patches and other lymphoid follicles.
A secretory IgA (sIgA) antibody which acts against the specific virulence determinant of infecting organisms plays a major role in mucosal immunity. In many cases, initial infection can be prevented by boosting the production of mucosal sIgA antibody which acts against the associated virulence determinant of the infecting organisms. The secretory IgA antibody is capable of preventing the initial interaction between the pathogens and mucosal surfaces by blocking their attachment and/or colonization, neutralizing surface-acting toxins, or by blocking their entry into host cells.
Edema disease of swine frequently develops in post-weaning pigs, due to infection with Escherichia coli. Although antibiotics are used for the prevention and treatment of edema disease of swine, it is an urgent matter to develop a preventive approach which could replace conventional methods using antibiotics, because of concerns for food safety and the emergence of resistant bacteria. However, there is no effective vaccine for edema disease at present. Escherichia coli diarrhea develops in young pigs during the lactation period and immediately after weaning. Although vaccines for use in the lactation period, designed to be administered to mother pigs, are commercially available, they cannot protect young pigs against diarrhea during the weaning period when maternal antibodies are starting to wear off. In addition, vaccines for preventing diarrhea which occurs during the weaning period are not commercially available. Thus, it is considered to be very useful to develop an orally administrable vaccine which is capable of preventing these diseases at the same time.
Patent Document 1 discloses the transformation of Arabidopsis thaliana cultured cells and a fragment of lettuce leaves in such a way that modified B subunit of edema disease toxin (Stx2eB) in which glycosylation is inhibited is transiently expressed. However, the effect of Stx2eB produced in the transformant when it is actually used as a vaccine has not been confirmed.
Patent Document 2 discloses a technique to link a vaccine antigen(s) for edema disease of swine and an antigen(s) for Escherichia coli diarrhea, to achieve a high accumulation of target proteins in a transgenic plant. However, in Patent Document 2, an issue that whether the above mentioned transgenic plant can be actually used as a vaccine or not remains unconfirmed. Further, Patent Document 2 is silent about the effects of: (1) the arrangement order in which a B subunit(s) of Escherichia coli heat-labile toxin (LTB) and a Stx2eB (s) are fused; and (2) the addition of an N-linked sugar chain to the LTB, on the level of antigen accumulation in plant cells, and on the performance as a vaccine.
Non-patent Document 1 discloses the expression and purification of a Stx2eB-LTB fusion antigen using recombinant Escherichia coli, and the evaluation of its performance. However, the performance evaluation of the fusion antigen when expressed in a plant such as strawberry and lettuce as a host is not described therein. Further, in Non-patent Document 1, the effect of the arrangement order in which LTB and Stx2eB are fused on the level of antigen accumulation in plant cells and on the performance as a vaccine has not been examined. In addition, although the effect of the Stx2eB-LTB fusion antigen to protect against Stx2e toxin has been confirmed, its effect in terms of protecting against LT toxin has not been evaluated. Moreover, Non-patent Document 1 describes the administration of the fusion antigen to an animal by injection (with the addition of an adjuvant), and it has not been confirmed whether or not the induction of immunity is possible by oral administration (without the addition of an adjuvant), which is an easier and more cost effective method.